Method for inducing a response in one or more targeted cells

ABSTRACT

A method and apparatus is disclosed for selectively identifying, and targeting with an energy beam, specific cells within a mixed cell population, for the purpose of inducing a response in the targeted cells. Two or more fluorescent labels can be attached to the targeted cells, and distinguished from one another by a color camera. By use of this system, one can monitor and affect cells having a desired phenotype. For example, targeted cells can not only be inactivated by an energy beam, but can also be monitored through fluorescent labeling for changes in cell morphology, ion transport or other cellular functions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/049,677, filed on Mar. 27, 1998, now U.S. Pat. No. 6,143,535, issuedNov. 7, 2000, the disclosure of which is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and apparatus for selectivelytargeting specific cells within a mixed population of living cells. Inparticular, this invention relates to methods and apparatus forselectively identifying, and individually targeting with an energy beam,specific cells within a mixed cell population to induce a response inthe targeted cells.

2. Description of the Related Art

The use of cellular therapies is growing rapidly, and is thereforebecoming an important therapeutic modality in the practice of medicine.Unlike other therapies, cellular therapies achieve a long-lasting, andoften permanent benefit through the use of living cells. Hematopoieticstem cell (HSC) (e.g., bone marrow or mobilized peripheral blood)transplantation is one example of a practiced, insurance-reimbursedcellular therapy. Many other cellular therapies are being developed,including immunotherapy for cancer and infectious diseases, chondrocytetherapy for cartilage defects, neuronal cell therapy forneurodegenerative diseases, and stem cell therapy for numerousindications. Many of these therapies require the removal of unwanted,detrimental cells for full efficacy to be realized.

Gene therapy is another active area of developing medicine that caninfluence the success of cellular therapy. Given the rapid advances inthe understanding of the human genome, it is likely that many genes willbe available for insertion into cells prior to transplantation intopatients. However, obtaining efficient targeted delivery of genes intospecific cells of interest has remained a difficult obstacle in thedevelopment of these therapies.

In the treatment of cancer, it has been found that high-dosechemotherapy and/or radiation therapy can be used to selectively killrapidly dividing cancer cells in the body. Unfortunately, several othercell types in the body are also rapidly dividing, and in fact, thedose-limiting toxicity for most anti-cancer therapies is the killing ofHSCs and progenitor cells in the bone marrow. HSC transplantation wasdeveloped as a therapy to rescue the hematopoietic system followinganti-cancer treatments. Upon infusion, the HSCs and progenitor cellswithin the transplant selectively home to the bone marrow and engraft.This process is monitored clinically through daily blood cell counts.Once blood counts return to acceptable levels, usually within 20 to 30days, the patient is considered engrafted and is released from thehospital.

HSC transplants have been traditionally performed with bone marrow, butmobilized peripheral blood (obtained via leukapheresis after growthfactor or low-dose chemotherapy administration) has recently become thepreferred source because it eliminates the need to harvest approximatelyone liter of bone marrow from the patient. In addition, HSCs frommobilized peripheral blood result in more rapid engraftment (8 to 15days), leading to less critical patient care and earlier discharge fromthe hospital. HSC transplantation has become an established therapy fortreating many diseases, such that over 45,000 procedures were performedworldwide in 1997.

HSC transplantation may be performed using either donor cells(allogeneic), or patient cells that have been harvested andcryopreserved prior to administration of high-dose anti-cancer therapy(autologous). Autologous transplants are widely used for treating avariety of diseases including breast cancer, Hodgkin's and non-Hodgkin'slymphomas, neuroblastoma, and multiple myeloma. The number of autologoustransplants currently outnumbers allogeneic transplants by approximatelya 2:1 ratio. This ratio is increasing further, mainly due tograft-versus-host disease (GVHD) complications associated withallogeneic transplants. One of the most significant problems withautologous transplants is the reintroduction of tumor cells to thepatient along with the HSCs, because these tumor cells contribute torelapse of the original disease.

As a tumor grows, tumor cells eventually leave the original tumor siteand migrate through the bloodstream to other locations in the body. Thisprocess, called tumor metastasis, results in the formation and growth ofsatellite tumors that greatly increase the severity of the disease. Thepresence of these metastatic tumor cells in the blood and other tissues,often including bone marrow, can create a significant problem forautologous transplantation. In fact, there is a very high probabilitythat metastatic tumor cells will contaminate the harvested HSCs that areto be returned to the patient following anti-cancer therapy.

The presence of contaminating tumor cells in autologous bone marrow andmobilized peripheral blood harvests has been confirmed in numerousscientific studies. Tumor cell contamination has been repeatedlyobserved in patients with T-cell lymphoma, non-Hodgkin's lymphoma,leukemia, neuroblastoma, lung cancer, breast cancer, etc. (Brugger, W.,Bross, K. J., Glatt, M., Weber, F., Mertelsmann, R., and Kanz, L.:Mobilization of tumor cells and hematopoietic progenitor cells intoperipheral blood of patients with solid tumors. Blood 83: 636-640, 1994;Gulati, S. C. and Acaba, L.: Rationale for purging in autologous stemcell transplantation. J.Hematotherapy 2: 467-471, 1993; Kvalheim, G.,Holte, H., Jakobsen, E., and Kvaloy, S.: Immunomagnetic purging oflymphoma cells from autografts. J.Hematotherapy 5: 561-562, 1996;Mapara, M. Y., Körner, I. J., Hildebrandt, M., Bargou, R., Krahl, D.,Reichardt, P., and Dörken, B.: Monitoring of tumor cell purging afterhighly efficient immunomagnetic selection of CD34 cells fromleukapheresis products in breast cancer patients: Comparison ofimmunocytochemical tumor cell staining and reversetranscriptase-polymerase chain reaction. Blood 89: 337-344, 1997;Paulus, U., Dreger, P., Viehmann, K., von Neuhoff, N., and Schmitz, N.:Purging peripheral blood progenitor cell grafts from lymphoma cells:Quantitative comparison of immunomagnetic CD34⁺ selection systems. StemCells 15: 297-304, 1997; Shpall, E. J. and Jones, R. B.: Release oftumor cells from bone marrow. Blood 83: 623-625, 1994; Vervoordeldonk,S. F., Merle, P. A., Behrendt, H., Steenbergen, E. J., van den Berg, H.,van Wering, E. R., von dem Borne, A. E. G., van der Schoot, C. E., vanLeeuwen, E. F., and Slaper-Cortenbach, I. C. M.: PCR-positivity inharvested bone marrow predicts relapse after transplantation withautologous purged bone marrow in children in second remission ofprecursor B-cell acute leukemia. Br.J.Haematol. 96: 395-402, 1997). Inevery study, all or nearly all of the patient samples analyzed werepositive for tumor contamination. The level of tumor cell burden inthese HSC harvests varied widely depending upon the type and stage ofdisease. Typical numbers indicate that tumor cells are present in therange of 3 to 3,000 tumor cells per million hematopoietic cells. Sincethe transplanted cell number is on the order of 10 billion hematopoieticcells, the total number of tumor cells in a transplant varies in therange of 30 thousand to 30 million. The reinfusion of this number oftumor cells in the HSC transplant following the patient's anti-cancertherapy is of considerable clinical concern. In fact, animal models haveshown that as few as 25 leukemia cells can establish a lethal tumor in50% of mice, and these numbers extrapolate to 3500 cells in humans(Gulati, Acaba 1993).

Recent landmark studies have unambiguously shown that reinfused tumorcells do indeed contribute to disease relapse in humans (Rill, D. R.,Santana, V. M., Roberts, W. M., Nilson, T., Bowman, L. C., Krance, R.A., Heslop, H. E., Moen, R. C., Ihle, J. N., and Brenner, M. K.: Directdemonstration that autologous bone marrow transplantation for solidtumors can return a multiplicity of tumorigenic cells. Blood 84:380-383, 1994). This was proven by genetically marking the harvestedcells prior to transplant, and then showing that the marker was detectedin resurgent tumor cells in those patients who relapsed with disease.These data have been confirmed by other investigators (Deisseroth, A.B., Zu, Z., Claxton, D., Hanania, E. G., Fu, S., Ellerson, D., Goldberg,L., Thomas, M., Janicek, K., Anderson, W. F., Hester, J., Korbling, M.,Durett, A., Moen, R., Berenson, R., Heimfeld, S., Hamer, J., Calver, L.,Tibbits, P., Talpaz, M., Kantarjiam, H., Champlin, R., and Reading, C.:Genetic marking shows that Ph⁺ cells present in autologous transplantsof chronic myelogenous leukemia (CML) contribute to relapse afterautologous bone marrow transplantation in CML. Blood 83: 3068-3076,1994), indicating that contaminating tumor cells in HSC transplantsrepresent a real threat to patients undergoing autologoustransplantation.

Subsequent detailed studies have now shown that the actual number oftumor cells reinfused in the transplant was correlated with the risk ofrelapse for acute lymphoblastic leukemia (Vervoordeldonk et al. 1997),non-Hodgkin's lymphoma (Sharp, J. G., Joshi, S. S., Armitage, J. O.,Bierman, P., Coccia, P. F., Harrington, D. S., Kessinger, A., Crouse, D.A., Mann, S. L., and Weisenburger, D. D.: Significance of detection ofoccult Non-Hodgkin's Lymphoma in histologically uninvolved bone marrowby a culture technique. Blood 79: 1074-1080, 1992; Sharp, J. G.,Kessinger, A., Mann, S., Crouse, D. A., Armitage, J. O., Bierman, P.,and Weisenburger, D. D.: Outcome of high-dose therapy and autologoustransplantation in non-Hodgkin's lymphoma based on the presence of tumorin the marrow or infused hematopoietic harvest. J.Clin.Oncol. 14:214-219, 1996), mantle cell lymphoma (Andersen, N. S., Donovan, J. W.,Borus, J. S., Poor, C. M., Neuberg, D., Aster, J. C., Nadler, L. M.,Freedman, A. S., and Gribben, J. G.: Failure of immunologic purging inmantle cell lymphoma assessed by polymerase chain reaction detection inminimal residual disease. Blood 90: 4212-4221, 1997), and breast cancer(Brockstein, B. E., Ross, A. A., Moss, T. J., Kahn, D. G.,Hollingsworth, K., and Williams, S. F.: Tumor cell contamination of bonemarrow harvest products: Clinical consequences in a cohort ofadvanced-stage breast cancer patients undergoing high-dose chemotherapy.J.Hematotherapy 5: 617-624, 1996; Fields, K. K., Elfenbein, G. J.,Trudeau, W. L., Perkins, J. B., Janssen, W. E., and Moscinski, L. C.:Clinical significance of bone marrow metastases as detected using thepolymerase chain reaction in patients with breast cancer undergoinghigh-dose chemotherapy and autologous bone marrow transplantation.J.Clin.Oncol. 14: 1868-1876, 1996; Schulze, R., Schulze, M., Wischnik,A., Ehnle, S., Doukas, K., Behr, W., Ehret, W., and Schlimok, G.: Tumorcell contamination of peripheral blood stem cell transplants and bonemarrow in high-risk breast cancer patients. Bone Marrow Transplant. 19:1223-1228, 1997; Vannucchi, A. M., Bosi, A., Glinz, S., Pacini, P.,Linari, S., Saccardi, R., Alterini, R., Rigacci, L., Guidi, S.,Lombarkini, L., Longo, G., Mariani, M. P., and Rossi-Ferrini, P.:Evaluation of breast tumour cell contamination in the bone marrow andleukapheresis collections by RT-PCR for cytokeratin-19 mRNA.Br.J.Haematol. 103: 610-617, 1998; Vredenburgh, J. J., Silva, O.,Broadwater, G., Berry, D., DeSombre, K., Tyer, C., Petros, W. P.,Peters, W. P., and Bast, J., R. C.: The significance of tumorcontamination in the bone marrow from high-risk primary breast cancerpatients treated with high-dose chemotherapy and hematopoietic support.Biol. Blood Marrow Transplant. 3: 91-97, 1997). One of these studieswent even further, showing that the number of tumor cells infused wasinversely correlated with the elapsed time to relapse (Vredenburgh etal. 1997). These data suggest that reducing the number of tumor cells inthe transplant will lead to better outcomes for the patient.

In fact, one clinical study of NHL purging in 114 patients showed thatdisease-free survival (after a median 2-year follow-up) wassubstantially higher (93%) in the subset of patients that had alldetectable tumor cells purged prior to transplant, as compared withthose in which purging was unsuccessful (54%) (Gribben, J. G., Freedman,A. S., Neuberg, D., Roy, D. C., Blake, K. W., Woo, S. D., Grossbard, M.L., Rabinowe, S. N., Coral, F., Freeman, G. J., Ritz, J., and Nadler, L.M.: Immunologic purging of marrow assessed by PCR before autologous bonemarrow transplantation for B-cell lymphoma. N.E.J.Med. 325: 1525-1533,1991). In a recent update of this study, eight-year freedom-from-relapsewas shown to be 83% in the subset of patients that had all detectabletumor cells purged, as compared to 19% in patients where purging wasunsuccessful (Freedman, A. S., Neuberg, D., Mauch, P., Soiffer, R. J.,Anderson, K. C., Fisher, D. C., Schlossman, R., Alyea, E. P., Takvorian,T., Jallow, H., Kuhlman, C., Ritz, J., Nadler, L. M., and Gribben, J.G.: Long-term follow-up of autologous bone marrow transplantation inpatients with relapsed follicular lymphoma. Blood 94: 3325-3333, 1999).Therefore, the actual number of tumor cells in an HSC transplant, andthe ability to reliably purge them, are of significant and growingimportance in the delivery of HSC transplantation therapies for cancerpatients.

Due to the known risk of tumor cell contamination in autologoustransplantation, a number of methods have been proposed for removingcontaminating tumor cells from harvested HSC populations. The basicprinciple underlying all purging methods is to remove or kill tumorcells while preserving the HSCs that are needed for hematopoieticreconstitution in the patient.

One method based on relatively non-specific adhesion differences ofhematopoietic cells in deep bed filtration has been described by Dooley,et al. (Dooley, D. C., Xiao, M., Wickramasinghe, S. R., Oppenlander, B.K., and Castino, F.: A novel inexpensive technique for the removal ofbreast cancer cells from mobilized peripheral blood stem cell products.Blood 88: 252a, 1996). An elutriation method based on relativelynon-specific cell size and density differences has been described byWagner, et al. (Wagner, J. E., Collins, D., Fuller, S., Schain, L. R.,Berson, A. E., Almici, C., Hall, M. A., Chen, K. E., Okarma, T. B., andLebkowski, J. S.: Isolation of small, primitive human hematopoitic stemcells: Distribution of cell surface cytokine receptors and growth inSCID-Hu mice. Blood 86: 512-523, 1995). Preferential killing of tumorcells by hyperthermia has been described by Higuchi, et al. (Higuchi,W., Moriyama, Y., Kishi, K., Koike, T., Shibata, A., Shinada, S., Tada,I., and Miura, A.: Hematopoietic recovery in a patient with acutelymphoblastic leukemia after an autologous marrow graft purged bycombined hyperthermia and interferon in vitro. Bone Marrow Transplant.7: 163-166, 1991). These relatively non-specific methods reduce thenumber of tumor cells present, but a significant number are known toremain.

In another method, cytotoxic agents, such as4-hydroxy-peroxy-cyclophosphamide (4-HC), were used to preferentiallykill tumor cells in populations containing HSCs (Bird, J. M., Luger, S.,Mangan, P., Edelstein, M., Silberstein, L., Powlis, W., Ball, J.,Schultz, D. J., and Stadtmauer, E. A.: 4-Hydroperoxycyclophosphamidepurged autologous bone marrow transplantation in non-Hodgkin's lymphomapatients at high risk of none marrow involvement. BMT 18: 309-313,1996). Unfortunately, collateral damage to normal HSCs was so severethat patient engraftment was delayed by as much as 59 days.

Another method employed preferential killing of tumor cells by exposingall cells to photoradiation in the presence of a light-sensitizing agent(e.g. merocyanide) (Gulliya, K. S. and Pervaiz, S.: Elimination ofclonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laserphotoradiation therapy: Implications for autologous bone marrow purging.Blood 73: 1059-1065, 1989). Although more tumor cells were killed thanhematopoietic cells, some tumor cells still remained, and HSC damage wassignificant.

Another method for removing tumor cells from populations ofhematopoietic cells involved immunoconjugating a toxic agent to anantibody having specificity for the tumor cells. In this system,antibodies were bound to chemotoxic agents, toxins, or radionucleidesand then contacted with the total cell population. Unfortunately, notall of the tumor cells were killed by this treatment (Gribben et al.1991; Robertson, M. J., Soiffer, R. J., Freedman, A. S., Rabinowe, S.L., Anderson, K. C., Ervin, T. J., Murray, C., Dear, K., Griffin, J. D.,Nadler, L. M., and Ritz, J.: Human bone marrow depleted of CD33-positivecells mediates delayed but durable reconstitution of hematopoiesis:Clinical trial of MY9 monoclonal antibody-purged autografts for thetreatment of acute myeloid leukemia. Blood 79: 2229-2236, 1992).

Some companies and physicians have attempted to purge malignant cellsfrom populations of non-tumor cells using an immunoaffinity bead-basedselection. In this procedure, the total cell population is contacted byimmunoaffinity beads. For example, a first (positive) CD34-selectionprocedure enriches HSCs from the tumor cell-containing hematopoieticcell mixture. In some instances, a second (negative) immunoaffinitybead-based selection is also performed using anti-tumor oranti-epithelial cell antibodies attached to the beads. Although theseprocedures enrich HSCs and reduce tumor cell numbers, tumor cells canstill be detected in the final product (Mapara, M. Y., Korner, I. J.,Lentzsch, S., Krahl, D., Reichardt, P., and Dorken, B.: Combinedpositive/negative purging and transplantation of peripheral bloodprogenitor cell autografts in breast cancer patients: A pilot study.Exp.Hematol. 27: 169-175, 1999).

In another protocol, Clarke et al. disclosed the use ofadenovirus-mediated transfer of suicide genes to selectively kill tumorcells (Clarke, M. F., Apel, I. J., Benedict, M. A., Eipers, P. G.,Sumantran, V., Gonzalez-Garcia, M., Doedens, M., Fukunaga, N., Davidson,B., Dick, J. E., Minn, A. J., Boise, L. H., Thompson, C. B., Wicha, M.,and Nunez, G.: A recombinant bcl-x_(S) adenovirus selectively inducesapoptosis in cancer cells but not in normal bone marrow cells. PNAS 92:11024-11028, 1995). However, it is well known that virus-mediated genetransfer is far less than 100% efficient, which would result in asignificant number of tumor cells being unaffected by the protocol.

Yet another method utilized fluorescence-activated cell sorting (FACS)to sort HSCs from tumor cells (Tricot, G., Gazitt, Y., Jagannath, S.,Vesole, D., Reading, C. L., Juttner, C. A., Hoffman, R., and Barlogie,B.: CD34⁺Thy⁺lin⁻ peripheral blood stem cells (PBSC) effect timelytrilineage engraftment in multiple myeloma (MM). Blood 86: 293a-0,1995). As is known, flow cytometry sorts cells one at a time andphysically separates one population of cells from a mixture of cellsbased upon cell surface markers and physical characteristics. However,it has been shown that using FACS to separate large cell populations forclinical applications is not advantageous because the process is slow,the cell yields can be very low, and purity greater than ˜98% is rarelyachieved.

Another method utilizing a flow cytometer is described in U.S. Pat. No.4,395,397 to Shapiro. In the Shapiro method, labeled cells are placed ina flow cytometer, and a downstream laser beam is used to kill thelabeled cells in the flowing stream after they pass by the detector andare recognized as being labeled by an electronic system. This methodsuffers from a number of disadvantages. Firstly, once an unwanted cellhas passed through the detector/laser region there is no way to checkthat destruction has been completed successfully. If a tumor cell evadesdestruction it will inevitably be reintroduced into the patient.Secondly, the focal spot diameter of the laser beam is of necessitygreater than the liquid stream cross section. Accordingly, many of theHSCs in the region of an unwanted cell will also be destroyed by thelaser beam. Also, as described above, flow cytometric techniques do notprovide pure samples of unlabeled cells due to the random and dynamicnature of the heterogeneous cell mixture that is flowing in afast-moving (1-20 m/sec) stream of liquid.

Another method that utilizes laser technology is described in U.S. Pat.No. 4,629,687 to Schindler, et al. In this method, anchorage-dependentcells are grown on a movable surface. A small laser beam spot is scannedacross the moving surface to illuminate cells one at a time and theinformation is then recorded. The same laser is then switched to alethal power level, and the beam is swept over the surface in all areasexcept where a cell of interest was recorded during the illuminationstep. Unfortunately, this method is slow and only will work on cellsthat can adhere to a surface.

A still further method that utilizes laser technology is described inU.S. Pat. No. 5,035,693 to Kratzer. In this method, cells are placed ona moving belt and a small laser beam spot is scanned across the beltsurface. When a particular cell radiates in response to the illuminatinglaser spot, the same laser is quickly switched to a high power settingin order to kill the cell in a near simultaneous manner before the laserhas moved appreciably away from that cell. However, this system has manyof the same disadvantages as the Shapiro method. For example, becausethe scanner is continuously moving during the imaging and killingprocess, the system is highly-dynamic, and therefore less stable andless accurate than a static system. Also, because the cells are movingon a belt past the detector in one direction, the method is notreversible. Thus, if a single tumor cell escapes detection, it will bereintroduced into the patient.

Others have used a small laser beam spot to dynamically scan over asurface to illuminate cells. For example, U.S. Pat. No. 4,284,897 toSawamura et al. describes the use of galvanometric mirrors to scan asmall laser beam spot in a standard microscope to illuminate fluorescentcells. U.S. Pat. No. 5,381,224 to Dixon et al. describes imaging ofmacroscopic specimens through the use of a laser beam spot that israster-scanned with galvanometric mirrors through an F-theta scanninglens. In U.S. Pat. Nos. 5,646,411, 5,672,880, and 5,719,391 to Kain,scanning of a small laser spot with galvanometers through an F-thetalens is described. All of these imaging methods dynamically illuminate asmall point that is moved over the surface to be imaged. In some cases,the surface being scanned also moves during imaging.

Similar methods of scanning a small laser spot have been described forpurposes other than imaging of cells. For example, U.S. Pat. No.4,532,402 to Overbeck describes the use of galvanometers to move a smalllaser beam spot over a semiconductor surface for repair of an integratedcircuit. Similarly, U.S. Pat. No. 5,690,846 to Okada et al. describeslaser processing by using mirrors to move a small laser spot through anF-theta scanning lens. U.S. Pat. No. 5,296,963 to Murakami et al.describes the use of galvanometric mirrors to scan a small laser beamspot in a standard inverted microscope to puncture cells for insertionof genetic matter.

Yet another method of scanning a biological specimen is described inU.S. Pat. No. 5,932,872 to Price. This method uses a plurality ofdetectors to simultaneously capture images at a plurality of focalplanes from a constantly moving surface. The resultant images were usedto choose the best-focus image in real-time in order to generate athree-dimensional volumetric image of a specimen.

The majority of methods described above are based on administering atumor cell-removal or tumor cell-killing strategy to the entireharvested cell population as a whole. In flow cytometry, cells aresorted on a single cell basis to physically separate the unwanted tumorcells from HSCs. While each of these methods has been shown to reducetumor cell numbers in HSC transplants, none has demonstrated the abilityto remove or kill all detectable tumor cells. In fact, the majority ofpatient transplants still contain detectable tumor cells after thesepurging techniques are used. Approximately 30 to 30,000 tumor cells pertransplant still remain, even after multiple-step purging procedures(Gazitt, Y., Reading, C. C., Hoffman, R., Wickrema, A., Vesole, D. H.,Jagannath, S., Condino, J., Lee, B., Barlogie, B., and Tricot, G.:Purified CD34⁺lin⁻Thy⁺ stem cells do not contain clonal myeloma cells.Blood 86: 381-389, 1995; Gribben et al. 1991; Mapara et al. 1997; Pauluset al. 1997).

Further, all of these methods result in some degree of HSC loss ordamage, which can significantly impact the success of the HSC transplantby delaying patient engraftment. In summary, existing technologies forseparating one cell population from another are inadequate, and thereexists a great unmet clinical need for novel approaches that caneffectively separate and treat one selected cell population within asecond cell population. The method and apparatus described hereinfulfills this need.

SUMMARY OF THE INVENTION

This invention provides a high-speed method and apparatus forselectively identifying, and individually targeting with an energy beam,specific cells within a mixed cell population of cells for the purposeof inducing a response in the targeted cells. Using the apparatus of thepresent invention, every detectable target cell in a mixed cellpopulation can be specifically identified and targeted, withoutsubstantially affecting cells that are not being targeted.

Specific cells are identified with the disclosed invention using severalapproaches. One embodiment includes a non-destructive labeling method sothat all of the target cells are substantially distinguishable from thenon-target cells in the specimen. In this embodiment, a labeled antibodycan be used to specifically mark each target cell, yet not marknon-target cells. The labeled cells are then identified within the cellmixture. A narrow energy beam is thereafter focused on the first of thetargeted cells to achieve a desired response. The next of the targetedcells is then irradiated, and so on until every targeted cell has beenirradiated.

In another embodiment, an antibody that selectively binds to non-targetcells, but not to target cells, is used to identify the target cells bythe absence of the label, and target cells are thereafter individuallytargeted with the energy beam.

To provide even greater flexibility in the ability to distinguish targetcells from non-target cells, combinations of two or more labels, eachwith different fluorescent properties, can be used. For example, oneantibody labeled with phycoerythrin (PE) and another antibody labeledwith Texas Red® could be used to identify target cells that express one,both, or neither of the antigens recognized by the antibodies. Oneskilled in the art could propose the use of many multi-color labelingapproaches to identify specific cell subpopulations within a complexmixture of cells.

The nature of the response that is induced by the energy beam isdependent upon the nature of the energy beam. Responses that can beinduced with an energy beam include necrosis, apoptosis, optoporation(to allow entry of a substance that is present in the surroundingmedium, including genetic material), cell lysis, cell motion (lasertweezers), cutting of cell components (laser scissors), activation of aphotosensitive substance, excitation of a fluorescent reagent, etc.

One embodiment of the invention is an apparatus for selectively inducinga response in one or more targeted cells within a biological specimenthat includes: an illumination source for illuminating a frame of cellsin the biological specimen with a first wavelength of light; an imagecapture system that captures one or more images of the frame of cells;first commands for determining the locations of the one or more targetedcells within the frame of cells; an energy source that emits an energybeam sufficient to induce the response in at least one of the one ormore targeted cells within the frame of cells; and second commands forsteering the energy beam to the locations of the one or more targetedcells.

Still another embodiment of the invention is an apparatus forselectively inducing a response in one or more targeted cells within abiological specimen. This embodiment includes a first illuminationsource for illuminating a frame of cells in the biological specimen witha first wavelength of light; a second illumination source forilluminating a frame of cells in the biological specimen with a secondwavelength of light; an image capture system that captures one or moreimages of the frame of cells; first commands for determining thelocations of the one or more targeted cells within the frame of cells;an energy source that emits an energy beam sufficient to induce theresponse in at least one of the one or more targeted cells within theframe of cells; and second commands for steering the energy beam to thelocations of the one or more targeted cells.

A still further embodiment is an apparatus for selectively inducing afirst response in a first targeted cell population and a second responsein a second targeted cell population within a biological specimen,providing: a means for illuminating a frame of cells in the biologicalspecimen with one or more wavelengths of light; an image capture systemthat captures one or more images of the first targeted cell populationand the second targeted cell population; first commands for determiningthe coordinates of one or more cells within the first targeted cellpopulation and one or more cells within the second targeted cellpopulation by reference to the captured one or more images; an energysource that emits an energy beam; and second commands for steering theenergy beam to the coordinates.

One additional embodiment is an apparatus for monitoring the status oflabeled cells within a biological sample. This embodiment includes: anillumination source for illuminating a population of cells in thebiological sample so that the labeled cells are distinguishable fromnon-labeled cells; an image capture system that captures one or moreimages of the illuminated population of cells; first commands fordetermining the locations of labeled cells in the one or more images; amemory for storing the locations of the labeled cells in the biologicalsample; an energy source that emits a first energy beam to illuminate afirst labeled cell; and a detector for measuring the fluorescence of thefirst labeled cell in response to illumination by the energy beam.

A further embodiment is a method for selectively inducing a response inone or more targeted cells within a biological specimen that includes:illuminating a frame of cells in the biological specimen with a firstwavelength of light, wherein the biological specimen has been treatedwith one or more labels that, when excited by the first wavelength oflight, emit energy at a one or more different wavelengths; capturing animage of the frame of cells; determining the locations of one or moretargeted cells within the image by reference to cells that are activatedby the first wavelength of light; and steering an energy beam to thelocations of the one or more targeted cells, wherein the energy beam issufficient to induce a response in at least one of the one or moretargeted cells.

Yet a further embodiment is a method for inducing a first response in afirst targeted population of cells and a second response in a secondtargeted population of cells, comprising: illuminating the firsttargeted population of cells and the second targeted population of cellswith a first wavelength of light; capturing an image comprising theilluminated first targeted population of cells and the illuminatedsecond targeted population of cells; determining the locations of thefirst targeted population of cells by reference to the image;determining the locations of the second targeted population of cells byreference to the image; and steering an energy beam to the locations ofthe first targeted population of cells and the second targetedpopulation of cells in order to induce the first response in the firsttargeted population of cells and the second response in the secondtargeted population of cells.

A large number of commercially important research and clinicalapplications can be envisioned for such an apparatus, examples of whichare presented below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of a cell treatmentapparatus and illustrates the outer design of the housing and display.

FIG. 2 is a perspective view of one embodiment of a cell treatmentapparatus with the outer housing removed and the inner componentsillustrated.

FIG. 3 is a block diagram of the optical subassembly design within oneembodiment of a cell treatment apparatus.

FIG. 4 is a perspective view of one embodiment of an optical subassemblywithin one embodiment of a cell treatment apparatus.

FIG. 5 is a side view of one embodiment of an optical subassembly thatillustrates the arrangement of the scanning lens and the movable stage.

FIG. 6 is a bottom perspective view of one embodiment of an opticalsubassembly.

FIG. 7 is a top perspective view of the movable stage of the celltreatment apparatus.

DETAILED DESCRIPTION

A method and apparatus is described for selectively identifying, andindividually targeting with an energy beam, specific cells within amixed cell population for the purpose of inducing a response in thetargeted cells. Generally, the method first employs a label or labelsthat act as markers to identify and locate specific cells within a cellmixture.

Use of one label leads to the identification of at least two cellsubpopulations; cells expressing the label, and cells lacking the label.Additional subpopulations can be further identified by the degree oflabel expression. For example, cells with bright expression or dimexpression can be distinguished. In addition, one can detect otherobservable cell parameters, such cell shape or size. Use of additionallabels that are excited, or emit light, at different wavelengths, allowsgreater flexibility for identifying many cell subpopulations within themixture.

Many such labels are available for cell identification. For example,monoclonal antibodies that are directly or indirectly tagged with afluorochrome can be used as specific labels. Other examples of cellsurface binding labels include non-antibody proteins, lectins,carbohydrates, or short peptides with selective cell binding capacity.Membrane intercalating dyes, such as PKH-2 and PKH-26 (Sigma, St. Louis,Mo.), also serve as useful distinguishing labels indicating mitotichistory of a cell. Many membrane-permeable reagents are also availableto distinguish living cells from one another based upon selectedcriteria. For example, phalloidin indicates membrane integrity,tetramethyl rhodamine methyl ester (TMRM) indicates mitochondrialtransmembrane potential, monochlorobimane indicates glutathionereductive stage, carboxymethyl fluorescein diacetate (CMFDA) indicatesthiol activity, carboxyfluorescein diacetate indicates intracellular pH,fura-2 indicates intracellular Ca²⁺ level, and5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo carbocyanineiodide (JC-1) indicates membrane potential. Cell viability can beassessed by the use of fluorescent SYTO 13 or YO PRO reagents (MolecularProbes, Eugene, Oreg.). Similarly, a fluorescently-tagged genetic probe(DNA or RNA) could be used to label cells which carry a gene ofinterest, or express a gene of interest. Further, cell cycle status canbe assessed through the use of Hoechst 33342 dye to label existing DNAcombined with bromodeoxyuridine (BrdU) to label newly synthesized DNA.It should be noted that the absence of a label can also be used foridentifying target cells.

It should be realized that embodiments of the invention include the useof several fluorochromes simultaneously. Preferably, each fluorochromeis excited by a single wavelength of light, but emits energy atdifferent light wavelengths. Thus, a color camera that can distinguishemissions at various light wavelengths can advantageously determinewhich cells in the population have one or more specific labels attached.

Accordingly, embodiments of the invention also include a camera thatimages a plurality of colors so that combinations of labels can beimaged simultaneously. For example, in one embodiment, a color CCDcamera is used to capture a color image of a population of cells.Because different cells within a population can be targeted by compoundsthat fluoresce at different wavelengths of light, images can be gatheredthat distinguish, on the basis of fluorescence wavelength, various celltypes in a mixed population. For example, the identity of a cancer cellcan be confirmed by its binding to a plurality of compounds that, whenexcited by an illumination laser, fluoresce at two differentwavelengths. A camera that detects both fluorescent molecules is able toconfirm the identity of the cancer cell more reliably than if the systemonly relied on a single identifying compound. It should be understoodthat each fluorescent compound might be excited by a differentwavelength of light. Thus, embodiments of the system includeillumination lasers that emit light at different wavelengths of light.Other embodiments of the system include two or more illumination lasersthat each emit light of a chosen wavelength.

After target cells are identified by reference to the one or morelabels, an energy beam, such as from a laser, collimated or focusednon-laser light, RF energy, accelerated particle, focused ultrasonicenergy, electron beam, or other radiation beam, delivers a targeted doseof energy. The amount of emitted energy is chosen to induce apre-determined response in each of the targeted cells, withoutsubstantially affecting non-targeted cells within the mixture. It isimportant to note that multiple target subpopulations can be identified,particularly with the use of multiple labels. Further, each targetsubpopulation can be irradiated with the energy beam in a differentmanner, inducing a different response in each target subpopulation.

FIG. 1 is an illustration of one embodiment of a cell treatmentapparatus 10. The cell treatment apparatus 10 includes a housing 15 thatstores the inner components of the apparatus. The housing includes lasersafety interlocks to ensure safety of the user, and also limitsinterference by external influences (e.g., ambient light, dust, etc.).Located on the upper portion of the housing 15 is a display unit 20 fordisplaying captured images of cell populations during treatment. Theseimages are captured by a camera, as will be discussed more specificallybelow. A keyboard 25 and mouse 30 are used to input data and control theapparatus 10. An access door 35 provides access to a movable stage thatholds a specimen container of cells undergoing treatment.

An interior view of the apparatus 10 is provided in FIG. 2. Asillustrated, the apparatus 10 provides an upper tray 200 and lower tray210 that hold the interior components of the apparatus. The upper tray200 includes a pair of intake filters 215A,B that filter ambient airbeing drawn into the interior of the apparatus 10. Below the access door35 is the optical subassembly (not shown). The optical subassembly ismounted to the upper tray 200 and is discussed in detail with regard toFIGS. 3-6.

On the lower tray 210 is a computer 225 which stores the softwareprograms, commands and instructions that run the apparatus 10. Inaddition, the computer 225 provides control signals to the treatmentapparatus through electrical signal connections for steering the laserto the appropriate spot on the specimen in order to treat the cells.

As illustrated, a series of power supplies 230A,B,C provide power to thevarious electrical components within the apparatus 10. In addition, anuninterruptable power supply 235 is incorporated to allow the apparatusto continue functioning through short external power interruptions.

FIG. 3 provides a layout of one embodiment of an optical subassemblydesign 300 within an embodiment of a cell treatment apparatus 10. Asillustrated, an illumination laser 305 provides a directed laser outputthat is used to excite one or more labels that are attached to targetedcells within the specimen. In this embodiment, the illumination laseremits light at, for example, a wavelength of 532 nm in order to exciteone or more specific labels. Of course lasers that illuminate light ofother wavelengths could also be used within the system. Once theillumination laser has generated a light beam, the light passes into ashutter 310 which controls the pulse length of the laser light.

After the illumination laser light passes through the shutter 310, itenters a ball lens 315 where it is focused into an SMA fiber opticconnector 320. After the illumination laser beam has entered the fiberoptic connector 320, it is transmitted through a fiber optic cable 325to an outlet 330. By passing the illumination beam through the fiberoptic cable 325, the illumination laser 305 can be positioned anywherewithin the treatment apparatus and thus is not limited to only beingpositioned within a direct light pathway to the optical components. Inone embodiment, the fiber optic cable 325 is connected to a vibratingmotor 327 for the purpose of mode scrambling and generating a moreuniform illumination spot.

After the light passes through the outlet 330, it is directed into aseries of condensing lenses in order to focus the beam to the properdiameter for illuminating one frame of cells. As used herein, one frameof cells is defined as the portion of the biological specimen that iscaptured within one frame image captured by the camera. This isdescribed more specifically below.

Accordingly, the illumination laser beam passes through a firstcondenser lens 335. In one embodiment, this first lens has a focallength of 4.6 mm. The light beam then passes through a second condenserlens 340 which, in one embodiment, provides a 100 mm focal length.Finally, the light beam passes into a third condenser lens 345, whichpreferably provides a 200 mm focal length. While the present inventionhas been described using specific condenser lenses, it should beapparent that other similar lens configurations that focus theillumination laser beam to an advantageous diameter would functionsimilarly. Thus, this invention is not limited to the specificimplementation of any particular condenser lens system.

Once the illumination laser beam passes through the third condenser lens345, it enters a cube beamsplitter 350 that transmits the 532 nmwavelength of light emanating from the illumination laser. Preferably,the cube beamsplitter 350 is a 25.4 mm square cube (Melles-Griot,Irvine, Calif.). However, other sizes are anticipated to functionsimilarly. In addition, a number of plate beamsplitters or pelliclebeamsplitters could be used in place of the cube beamsplitter 350 withno appreciable change in function.

Once the illumination laser light has been transmitted through the cubebeamsplitter 350, it reaches a long wave pass mirror 355 that reflectsthe 532 nm illumination laser light to a set of galvanometer mirrors 360that steer the illumination laser light, under computer control, througha scanning lens (Special Optics, Wharton, N.J.) 365 to the specimen (notshown). The galvanometer mirrors are controlled so that the illuminationlaser light is directed at the proper cell population (i.e. frame ofcells) for imaging.

The light from the illumination laser is of a wavelength that is usefulfor illuminating one or more labels in the specimen. In this embodiment,energy from a continuous wave 532 nm Nd:YAG frequency-doubled laser (B&WTek, Newark, Del.) reflects off the long wave pass mirror (CustomScientific, Phoenix, Ariz.) and excites fluorescent tags (labels) in thespecimen. In one embodiment, the fluorescent tag is phycoerythrin (PE).Alternatively, Alexa 532 (Molecular Probes, Eugene, Oreg.) can be usedas a fluorescent tag. Phycoerythrin and Alexa 532 have emission spectrawith peaks near 580 nm, so that the emitted fluorescent light from thespecimen is transmitted via the long wave pass mirror into the camera.The use of the filter in front of the camera blocks light that is notwithin the wavelength range of interest, thereby reducing the amount ofbackground light entering the camera.

The 532 nm illumination laser is further capable of exciting multiplefluorochromes that will emit energy at different wavelengths. Forexample, PE, Texas Red®, and CyChrome™ are all excited by a 532 nmlaser. However, they emit energy at 576 nm, 620 nm, and 670 nm,respectively. This difference in emitted wavelengths allows the signalfrom each fluorochrome to be distinguished from the others. In thiscase, the range of wavelengths transmitted by the filter 460 isexpanded. In addition, a camera that distinguishes various wavelengthsof light is used to capture the emitted light, so that cells which emitdifferent signals are distinguished by the computer. Alternatively, theemitted light from the targeted cells can be directed to threemonochromatic cameras, each having a filter to limit its vision to oneof the specific fluorochrome's emission wavelengths.

Yet another embodiment involves replacing the single fixed filter 460with a movable filter wheel that provides different filters that aremoved in and out of the optical pathway. In such an embodiment,fluorescent images of different wavelengths of light are captured atdifferent times during cell processing. The images are then analyzed andcorrelated by the computer, providing multicolor information about eachcell target or the cell population as a whole.

It is generally known that many other devices could be used in thismanner to illuminate the specimen, including, but not limited to, an arclamp (e.g., mercury, xenon, etc.) with or without filters, alight-emitting diode (LED), other types of lasers, etc. Advantages ofthis particular laser include high intensity, relatively efficient useof energy, compact size, and minimal heat generation. It is alsogenerally known that other fluorochromes with different excitation andemission spectra could be used in such an apparatus with the appropriateselection of illumination source, filters, and long and/or short wavepass mirrors. For example, Texas Red®, allophycocyanin (APC), andPharRed™ could all be excited with a 633 nm HeNe illumination laser,whereas fluoroisothiocyanate (FITC), PE, and CyChrome™ could all beexcited with a 488 nm Argon illumination laser.

One skilled in the art could propose many other optical layouts withvarious components in order to illuminate cells so that they emitfluorescent energy in multiple wavelengths. The illumination sourcesdescribed above can be used alone or in combination with other sourcesto provide a wide variety of illumination wavelengths within a singleinstrument, thereby allowing the use of many distinguishable labelssimultaneously.

In addition to the illumination laser 305, a treatment laser 400 ispresent to irradiate the targeted cells once they have been identifiedby image analysis. Of course, in one embodiment, the treatment inducesnecrosis of targeted cells within the cell population. As shown, thetreatment laser 400 outputs an energy beam of 523 nm that passes througha shutter 410. Although the exemplary laser outputs an energy beamhaving a 523 nm wavelength, other sources that generate energy at otherwavelengths are also within the scope of the present invention.

Once the treatment laser energy beam passes through the shutter 410, itenters a beam expander (Special Optics, Wharton, N.J.) 415 which adjuststhe diameter of the energy beam to an appropriate size at the plane ofthe specimen. Following the beam expander 415 is a half-wave plate 420which controls the polarization of the beam. The treatment laser energybeam is then reflected off a mirror 425 and enters the cube beamsplitter350. The treatment laser energy beam is reflected by 90° in the cubebeamsplitter 350, such that it is aligned with the exit pathway of theillumination laser light beam. Thus, the treatment laser energy beam andthe illumination laser light beam both exit the cube beamsplitter 350along the same light path. From the cube beamsplitter 350, the treatmentlaser beam reflects off the long wave pass mirror 355, is steered by thegalvanometers 360, thereafter enters the scanning lens 365, and finallyis focused upon a targeted cell within the specimen.

It should be noted that a small fraction of the illumination laser lightbeam passes through the long wave pass mirror 355 and enters a powermeter sensor (Gentec, Palo Alto, Calif.) 445. The fraction of the beamentering the power sensor 445 is used to calculate the level of poweremanating from the illumination laser 305. In an analogous fashion, asmall fraction of the treatment laser energy beam passes through thecube beamsplitter 350 and enters a second power meter sensor (Gentec,Palo Alto, Calif.) 446. The fraction of the beam entering the powersensor 446 is used to calculate the level of power emanating from thetreatment laser 400. The power meter sensors are electrically linked tothe computer system so that instructions/commands within the computersystem capture the power measurement and determine the amount of energythat was emitted from the treatment laser.

The energy beam from the treatment laser is of a wavelength that isuseful for achieving a response in the cells. In the example shown, apulsed 523 nm Nd:YLF frequency-doubled laser is used to heat a localizedvolume of fluid containing the targeted cell, such that it is induced todie within a pre-determined period of time. The mechanism of cell deathis dependent upon the actual temperature achieved in the cell, asreviewed by Niemz (Niemz, M. H.: Laser-tissue interactions: Fundamentalsand applications. Springer-Verlag, Berlin, 1996).

A Nd:YLF frequency-doubled, solid-state laser (Spectra-Physics, MountainView, Calif.) is used because of its stability, high repetition rate offiring, and long time of maintenance-free service. However, most cellculture fluids and cells are relatively transparent to light in thisgreen wavelength, and therefore a very high fluence of energy isrequired to achieve cell death. To significantly reduce the amount ofenergy required, and therefore the cost and size of the treatment laser,in one embodiment a dye is purposefully added to the cell culture toefficiently absorb the energy of the treatment laser in the specimen. Inthe example shown, the non-toxic dye FD&C red #40 (allura red) is usedto absorb the 523 nm energy from the treatment laser. However, oneskilled in the art could identify other laser/dye combinations thatwould result in efficient absorption of energy by the specimen. Forexample, a 633 nm HeNe laser's energy would be efficiently absorbed byFD&C green #3 (fast green FCF). Alternatively, a 488 nm Argon laser'senergy would be efficiently absorbed by FD&C yellow #5 (sunset yellowFCF), and a 1064 nm Nd:YAG laser's energy would be efficiently absorbedby Filtron (Gentex, Zeeland, Mich.) infrared absorbing dye. Through theuse of an energy absorbing dye, the amount of energy required to kill atargeted within a cell population cell can be reduced since more of thetreatment laser energy is absorbed in the presence of such a dye.

Another method of achieving thermal killing of cells without theaddition of a dye involves the use of an ultraviolet laser. Energy froma 355 nm Nd:YAG frequency-tripled laser will be absorbed by nucleicacids and proteins within the cell, resulting in very localized thermalheating and death.

Yet another method of achieving thermal killing of cells without theaddition of a dye involves the use of a near-infrared laser. Energy froma 2100 nm Ho:YAG laser or a 2940 nm Er:YAG laser will be absorbed bywater within the cell, resulting in thermal heating and death.

Although this embodiment describes the killing of cells via thermalheating by the energy beam, one skilled in the art would recognize thatother responses can also be induced in the cells by an energy beam,including photomechanical disruption, photodissociation, photoablation,and photochemical reactions. Also, a photosensitive substance (e.g.,hemtoporphyrin derivative, tin-etiopurpurin, lutetuim texaphyrin) withinthe cell mixture could be specifically activated in targeted cells byirradiation. Additionally, a small, transient pore could be made in thecell membrane to allow the entry of genetic or other material. Further,specific molecules in or on the cell, such as proteins or geneticmaterial, could be inactivated by the directed energy beam. Thesemechanisms of inducing a response in a targeted cell via the use ofelectromagnetic radiation directed at specific targeted cells are alsointended to be incorporated into the present invention.

As discussed above, multiple cell subpopulations can be identified withthe appropriate use of specific labels and illumination sources.Further, multiple cellular responses can be induced with the appropriateuse of treatment lasers and treatment substances added to the biologicalspecimen. By extension, the simultaneous identification and processingof different cell subpopulations (i.e. to induce different responses) inparallel is possible. For example, one cell subpopulation can beidentified and targeted for induction of necrosis, while another cellsubpopulation in the same specimen can be targeted for optoporation. Asthe process is carried out, both cell subpopulations are treated in theappropriate manner, under control of the computer.

In addition to the illumination laser 305 and treatment laser 400, theapparatus includes a camera 450 that captures images (i.e. frames) ofthe cell populations. As illustrated in FIG. 3, the camera 450 isfocused through a lens 455 and filter 460 in order to accurately recordan image of the cells without capturing stray background images. A stop462 is positioned between the filter 460 and mirror 355 in order toeliminate light that may enter the camera from angles not associatedwith the image from the specimen. The filter 460 is chosen to only allowpassage of light within a certain wavelength range. This wavelengthrange includes light that is emitted from the targeted cells uponexcitation by the illumination laser 305, as well as light from aback-light source 475.

The back-light source 475 is located above the specimen to provideback-illumination of the specimen at a wavelength different from thatprovided by the illumination laser 305. This LED generates light at 590nm, such that it can be transmitted through the long wave pass mirror tobe directed into the camera. This back-illumination is useful forimaging cells when there are no fluorescent targets within the framebeing imaged. An example of the utility of this back-light is its use inattaining proper focus of the system, even when there are onlyunstained, non-fluorescent cells in the frame. In one embodiment, theback-light is mounted on the underside of the access door 35 (FIG. 2).

Thus, as discussed above, the only light returned to the camera is fromwavelengths that are of interest in the specimen. Other wavelengths oflight do not pass through the filter 460, and thus do not becomerecorded by the camera 450. This provides a more reliable mechanism forcapturing images of only those cells of interest.

It should be noted that in this embodiment, the camera is acharge-coupled device (CCD) and transmits images back to the computersystem for processing. As will be described below, the computer systemdetermines the coordinates of the targeted cells in the specimen byreference to the image captured by the CCD camera.

Referring now to FIG. 4, a perspective view of an embodiment of anoptical subassembly is illustrated. As illustrated, the illuminationlaser 305 sends a light beam through the shutter 310 and ball lens 315to the SMA fiber optic connector 320. The light passes through the fiberoptic cable 325 and through the output 330 into the condenser lenses335, 340 and 345. The light then enters the cube beamsplitter 350 and istransmitted to the long wave pass mirror 355. From the long wave passmirror 355, the light beam enters the computer-controlled galvanometers360 and is then steered to the proper frame of cells in the specimenthrough the scanning lens 365.

As also illustrated in the perspective drawing of FIG. 4, the treatmentlaser 400 transmits energy through the shutter 410 and into the beamexpander 415. Energy from the treatment laser 400 passes through thebeam expander 415 and passes through the half-wave plate 420 beforehitting the fold mirror 425, entering the cube beamsplitter 350 where itis reflected 90° to the long wave pass mirror 355, from which it isreflected into the computer controlled galvanometer mirrors 360. Afterbeing steered by the galvanometer mirrors 360 through the scanning lens365, the laser energy beam strikes the proper location within the cellpopulation in order to induce a response in a particular targeted cell.

In order to accommodate a very large surface area of specimen to treat,the apparatus includes a movable stage that mechanically moves thespecimen container with respect to the scanning lens. Thus, once aspecific sub-population (i.e. field) of cells within the scanning lensfield-of-view has been treated, the movable stage brings anothersub-population of cells within the scanning lens field-of-view. Asillustrated in FIG. 5, a computer-controlled movable stage 500 holds aspecimen container (not shown) to be processed. The movable stage 500 ismoved by computer-controlled servo motors along two axes so that thespecimen container can be moved relative to the optical components ofthe instrument. The stage movement along a defined path is coordinatedwith other operations of the apparatus. In addition, specificcoordinates can be saved and recalled to allow return of the movablestage to positions of interest. Encoders on the x and y movement provideclosed-loop feedback control on stage position.

The flat-field (F-theta) scanning lens 365 is mounted below the movablestage. The scanning lens field-of-view comprises the portion of thespecimen that is presently positioned above the scanning lens by themovable stage 500. The lens 365 is mounted to a stepper motor thatallows the lens 365 to be automatically raised and lowered (along thez-axis) for the purpose of focusing the system.

As illustrated in FIGS. 4-6, below the scanning lens 365 are thegalvanometer-controlled steering mirrors 360 that deflectelectromagnetic energy along two perpendicular axes. Behind the steeringmirrors is the long wave pass mirror 355 that reflects electromagneticenergy of a wavelength shorter than 545 nm. Wavelengths longer than 545nm are passed through the long wave pass mirror, directed through thefilter 460, coupling lens 455, and into the CCD camera, therebyproducing an image of the appropriate size on the CCD sensor of thecamera 450 (See FIGS. 3 and 4). The magnification defined by thecombination of the scanning lens 365 and coupling lens 455 is chosen toreliably detect single cells while maximizing the area viewed in oneframe by the camera. Although a CCD camera (DVC, Austin, Tex.) isillustrated in this embodiment, the camera can be any type of detectoror image gathering equipment known to those skilled in the art. Theoptical subassembly of the apparatus is preferably mounted on avibration-isolated platform to provide stability during operation asillustrated in FIGS. 2 and 5.

Referring now to FIG. 7, a top view of the movable stage 500 isillustrated. As shown, a specimen container is mounted in the movablestage 500. The specimen container 505 rests on an upper axis nest plate510 that is designed to move in the forward/backward direction withrespect to the movable stage 500. A stepper motor (not shown) isconnected to the upper axis nest plate 510 and computer system so thatcommands from the computer cause forward/backward movement of thespecimen container 505.

The movable stage 500 is also connected to a timing belt 515 thatprovides side-to-side movement of the movable stage 500 along a pair ofbearing tracks 525A,B. The timing belt 515 attaches to a pulley (notshown) housed under a pulley cover 530. The pulley is connected to astepper motor 535 that drives the timing belt 515 to result inside-to-side movement of the movable stage 500. The stepper motor 535 iselectrically connected to the computer system so that commands withinthe computer system result in side-to-side movement of the movable stage500. A travel limit sensor 540 connects to the computer system andcauses an alert if the movable stage travels beyond a predeterminedlateral distance.

A pair of accelerometers 545A,B is preferably incorporated on thisplatform to register any excessive bumps or vibrations that mayinterfere with the apparatus operation. In addition, a two-axisinclinometer 550 is preferably incorporated on the movable stage toensure that the specimen container is level, thereby reducing thepossibility of gravity-induced motion in the specimen container.

The specimen chamber has a fan with ductwork to eliminate condensationon the specimen container, and a thermocouple to determine whether thespecimen chamber is within an acceptable temperature range. Additionalfans are provided to expel the heat generated by the electroniccomponents, and appropriate filters are used on the air intakes 215A,B.

The computer system 225 controls the operation and synchronization ofthe various pieces of electronic hardware described above. The computersystem can be any commercially available computer that can interfacewith the hardware. One example of such a computer system is an IntelPentium II-based computer running the Microsoft Windows® NT operatingsystem. Software is used to communicate with the various devices, andcontrol the operation in the manner that is described below.

When the apparatus is first initialized, the computer loads files fromthe hard drive into RAM for proper initialization of the apparatus. Anumber of built-in tests are automatically performed to ensure theapparatus is operating properly, and calibration routines are executedto calibrate the cell treatment apparatus. Upon successful completion ofthese routines, the user is prompted to enter information via thekeyboard and mouse regarding the procedure that is to be performed (e.g.patient name, ID number, etc.). Once the required information isentered, the user is prompted to open the access door 35 and load aspecimen onto the movable stage.

Once a specimen is in place on the movable stage and the door is closed,the computer passes a signal to the stage to move into a home position.The fan is initialized to begin warming and defogging of the specimen.During this time, cells within the specimen are allowed to settle to thebottom surface. In addition, during this time, the apparatus may runcommands that ensure that the specimen is properly loaded, and is withinthe focal range of the system optics. For example, specific markings onthe specimen container can be located and focused on by the system toensure that the scanning lens has been properly focused on the bottom ofthe specimen container. After a suitable time, the computer turns offthe fan to prevent excess vibrations during treatment, and celltreatment processing begins.

First, the computer instructs the movable stage to be positioned overthe scanning lens so that the first area (i.e. field) of the specimen tobe treated is directly in the scanning lens field-of-view. Thegalvanometer mirrors are instructed to move such that the center framewithin the field-of-view is imaged in the camera. As discussed below,the field imaged by the scanning lens is separated into a plurality offrames. Each frame is the proper size so that the cells within the frameare effectively imaged by the camera.

The back-light 475 is then activated in order to illuminate thefield-of-view so that it can be brought into focus by the scanning lens.Once the scanning lens has been properly focused upon the specimen, thecomputer system divides the field-of-view into a plurality of frames sothat each frame is analyzed separately by the camera. This methodologyallows the apparatus to process a plurality of frames within a largefield-of-view without moving the mechanical stage. Because thegalvanometers can move from one frame to the next very rapidly comparedto the mechanical steps involved in moving the stage, this methodresults is an extremely fast and efficient apparatus.

In one preferred embodiment, the apparatus described herein processes atleast 1, 3, 5, 7, or 9 square centimeters of a biological specimen perminute. In another embodiment, the apparatus described herein processesat least 1, 3, 5, 7, or 9 million cells of a biological specimen perminute. In one other embodiment, the apparatus can preferably induce aresponse in targeted cells at a rate of 250, 500, 1,000, 2,500, 5,000,or 10,000 cells per second.

Initially, an image of the frame at the center of the field-of-view iscaptured by the camera and stored to a memory in the computer.Instructions in the computer analyze the focus of the specimen bylooking at the size of, number of, and other object features in theimage. If necessary, the computer instructs the z-axis motor attached tothe scanning lens to raise or lower in order to achieve the best focus.The apparatus may iteratively analyze the image at several z-positionsuntil the best focus is achieved. The galvanometer-controlled mirrorsare then instructed to image a first frame, within the field-of-view, inthe camera. For example, the entire field-of-view might be divided into4, 9, 12, 18, 24 or more separate frames that will be individuallycaptured by the camera. Once the galvanometer mirrors are pointed to thefirst frame in the field-of-view, the shutter in front of theillumination laser is opened to illuminate the first frame through thegalvanometer mirrors and scanning lens. The camera captures an image ofany fluorescent emission from the specimen in the first frame of cells.Once the image has been acquired, the shutter in front of theillumination laser is closed and a software program (Epic, BuffaloGrove, Ill.) within the computer processes the image.

The power sensor 445 discussed above detects the level of light that wasemitted by the illumination laser, thereby allowing the computer tocalculate if it was adequate to illuminate the frame of cells. If not,another illumination and image capture sequence is performed. Repeatedfailure to sufficiently illuminate the specimen will result in an errorcondition that is communicated to the operator.

Shuttering of illumination light reduces undesirable heating andphotobleaching of the specimen and provides a more repeatablefluorescent signal. An image analysis algorithm is run to locate the x-ycentroid coordinates of all targeted cells in the frame by reference tofeatures in the captured image. If there are targets in the image, thecomputer calculates the two-dimensional coordinates of all targetlocations in relation to the movable stage position and field-of-view,and then positions the galvanometer-controlled mirrors to point to thelocation of the first target in the first frame of cells. It should benoted that only a single frame of cells within the field-of-view hasbeen captured and analyzed at this point. Thus, there should be arelatively small number of identified targets within this sub-populationof the specimen. Moreover, because the camera is pointed to a smallerpopulation of cells, a higher magnification is used so that each targetis imaged by many pixels within the CCD camera. For example, a clusterof 4, 8 16 or more pixels can be used to define a cell within the image.

Once the computer system has positioned the galvanometer controlledmirrors to point to the location of the first targeted cell within thefirst frame of cells, the treatment laser is fired for a brief intervalso that the first targeted cell is given an appropriate dose of energy.The power sensor 446 discussed above detects the level of energy thatwas emitted by the treatment laser, thereby allowing the computer tocalculate if it was adequate to induce a response in the targeted cell.If not sufficient, the treatment laser is fired at the same targetagain. If repeated shots do not deliver the required energy dose, anerror condition is communicated to the operator. These targeting,firing, and sensing steps are repeated by the computer for all targetsidentified in the captured frame.

Once all of the targets have been irradiated with the treatment laser inthe first frame of cells, the mirrors are then positioned to the secondframe of cells in the field-of-view, and the processing repeats at thepoint of frame illumination and camera imaging. This processingcontinues for all frames within the field-of-view above the scanninglens. When all of these frames have been processed, the computerinstructs the movable stage to move to the next field-of-view in thespecimen, and the process repeats at the back-light illumination andauto-focus step. Frames and fields-of-view are appropriately overlappedto reduce the possibility of inadvertently missing areas of thespecimen. Once the specimen has been fully processed, the operator issignaled to remove the specimen, and the apparatus is immediately readyfor the next specimen.

Although the text above describes the analysis of fluorescent images forlocating targets, one can easily imagine that the non-fluorescentback-light LED illumination images will be useful for locating othertypes of targets as well, even if they are unlabeled.

The advantage of using the galvanometer mirrors to control the imagingof successive frames and the irradiation of successive targets issignificant. One brand of galvanometer is the Cambridge Technology, Inc.model number 6860 (Cambridge, Mass.). This galvanometer can repositionvery accurately within a few milliseconds, making the processing oflarge areas and many targets possible within a reasonable amount oftime. In contrast, the movable stage is relatively slow, and istherefore used only to move specified areas of the specimen into thescanning lens field-of-view. Error signals continuously generated by thegalvanometer control boards are monitored by the computer to ensure thatthe mirrors are in position and stable before an image is captured, orbefore a target is fired upon, in a closed-loop fashion.

In the context of the present invention, the term “specimen” has a broadmeaning. It is intended to encompass any type of biological sampleplaced within the apparatus. The specimen may be enclosed by, orassociated with, a container to maintain the sterility and viability ofthe cells. Further, the specimen may incorporate, or be associated with,a cooling or heating apparatus to keep it above or below ambienttemperature during operation of the methods described herein. Thespecimen container, if one is used, must be compatible with the use ofthe illumination laser, back-light illuminator, and treatment laser,such that it transmits adequate energy without being substantiallydamaged itself.

One embodiment allows in vitro maintenance of the biological specimen inthe apparatus, such that it can be monitored and processed periodicallyin situ. This is accomplished by maintaining the specimen under astandard tissue culture environment of 37° C. In this manner, specificcells in the specimen are followed over time, periodically assessingtheir state, and/or periodically rendering treatment to those specificcells. The system includes software and computer memory storages forproviding the apparatus with the ability to record cell locations andthereafter direct the movable stage to return to those recordedlocations.

Of course, many variations of the above-described embodiments arepossible, including alternative methods for illuminating, imaging, andtargeting the cells. For example, movement of the specimen relative tothe scanning lens could be achieved by keeping the specimensubstantially stationary while the scanning lens is moved. Steering ofthe illumination beam, images, and energy beam could be achieved throughany controllable reflective or diffractive device, including prisms,piezo-electric tilt platforms, or acousto-optic deflectors.Additionally, the apparatus can image/process from either below or abovethe specimen. Because the apparatus is focused through a movablescanning lens, the illumination and energy beams can be directed todifferent focal planes along the z-axis. Thus, portions of the specimenthat are located at different vertical heights can be specificallyimaged and processed by the apparatus in a three-dimensional manner. Thesequence of the steps could also be altered without changing theprocess. For example, one might locate and store the coordinates of alltargets in the specimen, and then return to the targets to irradiatethem with energy one or more times over a period of time.

It should also be realized that more than one illumination laser couldadvantageously be provided within the cell treatment system. Forexample, various illumination lasers, each emitting light at a differentwavelength could be used to excite labels within the specimen. Cellshaving one or more excited labels would then be imaged by a camera thatwas capable of distinguishing the wavelength of light coming from eachlabel.

In another embodiment, multiple monochrome cameras could be used inplace of a single color camera to image a plurality of excited labels. Adifferent filter would advantageously be placed in front of eachmonochrome camera so that only light of one wavelength is allowed toenter the camera. The multiple images gathered by the camera array arethen processed by the computer system to determine which cell or cellshave been tagged with a particular label.

To optimally process the specimen, it should be placed on asubstantially flat surface so that a large portion of the specimenappears within a narrow range of focus, thereby reducing the need forrepeated auto-focus steps. The density of cells on this surface can, inprinciple, be at any value. However, the cell density should be as highas possible to minimize the total surface area required for theprocedure.

The following examples illustrate the use of the described method andapparatus in different applications.

EXAMPLE 1

Autologous HSC Transplantation

A patient with a B cell-derived metastatic tumor in need of anautologous HSC transplant is identified by a physician. As a first stepin the treatment, the patient undergoes a standard HSC harvestprocedure, resulting in collection of approximately 1×10¹⁰ hematopoieticcells with an unknown number of contaminating tumor cells. The harvestedcells are enriched for HSC by a commercial immunoaffinity column(Isolex® 300, Nexell Therapeutics, Irvine, Calif.) that selects forcells bearing the CD34 surface antigen, resulting in a population ofapproximately 3×10⁸ hematopoietic cells, with an unknown number of tumorcells. The mixed population is thereafter contacted with anti-B cellantibodies (directed against CD20 and CD22) that are conjugated tophycoerythrin, and anti-CD34 antibodies that are conjugated toCyChrome™.

The mixed cell population is then placed in a sterile specimen containeron a substantially flat surface near confluence, at approximately500,000 cells per square centimeter. The specimen is placed on themovable stage of the apparatus described above, and all detectable tumorcells are identified by the presence of the phycoerythrin tag andabsence of the CyChrome™ tag, and are then targeted with a lethal doseof energy from a treatment laser. The design of the apparatus allows theprocessing of a clinical-scale transplant specimen in under 4 hours. Thecells are recovered from the specimen container, washed, and thencryopreserved. Before the cells are reinfused, the patient is givenhigh-dose chemotherapy to destroy the tumor cells in the patient's body.Following this treatment, the processed cells are thawed at 37° C. andare given to the patient intraveneously. The patient subsequentlyrecovers with no remission of the original cancer.

EXAMPLE 2

Allogeneic HSC Transplantation

In another embodiment, the significant risk and severity ofgraft-versus-host disease in the allogeneic HSC transplant setting canbe combated. A patient is selected for an allogeneic transplant once asuitable donor is found. Cells are harvested from the selected donor asdescribed in the above example. In this case, the cell mixture iscontacted with phycoerythrin-labeled anti-CD3 T-cell antibodies andTexas Red®-labeled anti-CD6 antibodies. In addition, specificallo-reactive T-cell subsets are labeled using an activated T-cellmarker (e.g. CyChrome™-labeled anti-CD69 antibodies) in the presence ofallo-antigen. The cell population is processed by the apparatusdescribed herein, killing cells that are CD3⁺CD6⁺ and CD3⁺CD69⁺, butleaving all other cells, including CD3⁺CD6⁻ and CD3⁺CD69⁻ cells,unharmed. This type of control is advantageous, because administrationof too many T-cells increases the risk of graft-versus-host disease,whereas too few T-cells increases the risk of graft failure and the riskof losing of the known beneficial graft-versus-leukemia effect. Thepresent invention and methods are capable of precisely controlling thenumber and type of T-cells in an allogeneic transplant.

EXAMPLE 3

Tissue Engineering

In another application, the present apparatus is used to removecontaminating cells in inocula for tissue engineering applications. Cellcontamination problems exist in the establishment of primary cellcultures required for implementation of tissue engineering applications,as described by Langer and Vacanti (Langer, R. S. and Vacanti, J. P.:Tissue engineering: The challenges ahead. Sci.Am. 280: 86-89, 1999). Inparticular, chondrocyte therapies for cartilage defects are hampered byimpurities in the cell populations derived from cartilage biopsies.Accordingly, the present invention is used to specifically remove thesetypes of cells from the inocula.

For example, a cartilage biopsy is taken from a patient in need ofcartilage replacement. The specimen is then grown under conventionalconditions (Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C.,Isaksson, O., and Peterson, L.: Treatment of deep cartilage defects inthe knee with autologous chondrocyte transplantation. N.E.J.Med. 331:889-895, 1994). The culture is then stained with a specific label forany contaminating cells, such as fast-growing fibroblasts. The cellmixture is then placed within the apparatus described and the labeled,contaminating cells are targeted by the treatment laser, therebyallowing the slower growing chondrocytes to fully develop in culture.

EXAMPLE 4

Stem Cell Therapy

Yet another embodiment involves the use of embryonic stem cells to treata wide variety of diseases. Since embryonic stem cells areundifferentiated, they can be used to generate many types of tissue thatwould find use in transplantation, such as cardiomyocytes and neurons.However, undifferentiated embryonic stem cells that are implanted canalso lead to a jumble of cell types which form a type of tumor known asa teratoma (Pedersen, R. A.: Embryonic stem cells for medicine.Sci.Amer. 280: 68-73, 1999). Therefore, therapeutic use of tissuesderived from embryonic stem cells must include rigorous purification ofcells to ensure that only sufficiently differentiated cells areimplanted. The apparatus described herein is used to eliminateundifferentiated stem cells prior to implantation of embryonic stemcell-derived tissue in the patient.

EXAMPLE 5

Generation of Human Tumor Cell Cultures

In another embodiment, a tumor biopsy is removed from a cancer patientfor the purpose of initiating a culture of human tumor cells. However,the in vitro establishment of primary human tumor cell cultures frommany tumor types is complicated by the presence of contaminating primarycell populations that have superior in vitro growth characteristics overtumor cells, and the limited number of tumor cells obtainable from sucha sample. For example, contaminating fibroblasts represent a majorchallenge in establishing many cancer cell cultures. The disclosedapparatus is used to particularly label and destroy the contaminatingcells, while leaving the biopsied tumor cells intact. Accordingly, themore aggressive primary cells will not overtake and destroy the cancercell line. The apparatus described within allows purification of cellswith relatively high yield, which is particularly important inapplication where the starting cell number is limited. The high yield ofpurified cells from the disclosed apparatus provides a significantadvantage over other cell purification methods.

EXAMPLE 6

Generation of a Specific mRNA Expression Library

The specific expression pattern of genes within different cellpopulations is of great interest to many researchers, and many studieshave been performed to isolate and create libraries of expressed genesfor different cell types. For example, knowing which genes are expressedin tumor cells versus normal cells is of great potential value (Cossman,J. C., Annunziata, C. M., Barash, S., Staudt, L., Dillon, P., He, W.-W.,Ricciardi-Castognoli, P., Rosen, C. A., and Carter, K. C.:Reed-Sternberg cell genome expression supports a B-cell lineage. Blood94: 411-416, 1999). Due to the amplification methods used to generatesuch libraries (e.g. PCR), even a small number of contaminating cellswill result in an inaccurate expression library (Cossman et al. 1999;Schutze, K. and Lahr, G.: Identification of expressed genes bylaser-mediated manipulation of single cells. Nature Biotechnol. 16:737-742, 1998). One approach to overcome this problem is the use oflaser capture microdissection (LCM), in which a single cell is used toprovide the starting genetic material for amplification (Schutze, Lahr1998). Unfortunately, gene expression in single cells is somewhatstochastic, and may be biased by the specific state of that individualcell at the time of analysis (Cossman et al. 1999). Therefore, accuratepurification of a significant cell number prior to extraction of mRNAwould enable the generation of a highly accurate expression library, onethat is representative of the cell population being studied, withoutbiases due to single cell expression or expression by contaminatingcells. The methods and apparatus described in this invention can be usedto purify cell populations with high yield so that no contaminatingcells are present during an RNA extraction procedure.

Within a human prostate tumor, there are multiple cell types, each ofwhich lead to metastatic disease. To understand the basis of prostatecancer and its progression, the tools of genomics such as DNA sequencingof normal prostate and cancer prostate cDNAs (expressed sequence tags,or ESTs) and DNA array comparison of normal and cancerous tissues havebeen used to investigate the distinct patterns of gene expression innormal prostate and in different prostate tumors. It has been shown thatdifferent prostate cell subpopulations with different surface antigenshave different mRNA expression profiles. Using the apparatus disclosedwithin, primary human prostate tumors are purified for specific cellsubpopulations to investigate their gene expression. For example, fourpopulations of primary prostate cells are purified for mRNA analysis bybinding antibodies against specific cell markers to new cell surfaces.The purified populations are: CD44⁺CD13⁻ normal basal, CD44⁺CD13⁺ basalcell-like cancer, CD57⁺CD13⁺ normal luminal, and CD57⁺CD13⁻ luminalcell-like cancer epithelial cells. Understanding the nature of eachsubpopulation, and its relation to disease onset and progression, couldlead to new diagnostic and therapeutic approaches in the treatment ofthe disease.

EXAMPLE 7

Transfection, Monitoring, and Purification of a Specific Cell Population

Many research and clinical gene therapy applications are hampered by theinability to transfect an adequate number of a desired cell type withouttransfecting other cells that are present. The method of the presentinvention would allow selective targeting of cells to be transfectedwithin a mixture of cells. By generating a photomechanical shock wave ator near a cell membrane with a targeted energy source, a transient porecan be formed, through which genetic (or other) material can enter thecell. This method of gene transfer has been called optoporation(Palumbo, G., Caruso, M., Crescenzi, E., Tecce, M. F., Roberti, G., andColasanti, A.: Targeted gene transfer in eukaryotic cells bydye-assisted laser optoporation. J.Photochem.Photobiol. 36: 41-46,1996). The apparatus described above can achieve selective optoporationon only the cells of interest in a rapid, automated, targeted manner.

For example, bone marrow cells are plated in a specimen container havinga solution containing plasmid DNA to be transfected. The plasmid DNAencodes a therapeutic gene (e.g. MDR), as well as a marker gene (e.g.green fluorescent protein). PE-labeled antibodies (anti-CD34) havingspecificity for stem cells are added into the medium and bind to thestem cells. The specimen container is placed within the cell processingapparatus and a first treatment laser is targeted to any cells thatbecome fluorescent under the illumination laser light, therebyfacilitating transfection of DNA specifically into the targeted cells.The cells are maintained in situ at 37° C., all targeted cells beingperiodically analyzed for expression of green fluorescent protein toindicate successful transfection with the plasmid DNA. After 48 hours,all cells not expressing green fluorescent protein are eliminated with asecond treatment laser, thereby yielding a pure population of stem cellsexpressing the transfected genes.

EXAMPLE 8

Selection of Desirable Clones in a Biotechnology Application

In many biotechnology processes where cell lines are used to generate avaluable product, it is desirable to derive clones that are veryefficient in producing the product. This selection of clones is oftencarried out manually, by inspecting a large number of clones that havebeen isolated in some manner. The present invention would allow rapid,automated inspection and selection of desirable clones for production ofa particular product. For example, hybridoma cells that are producingthe greatest amounts of antibody can be identified by a fluorescentlabel directed against the F_(c) region. Cells with no or dimfluorescent labeling are targeted by the treatment laser for killing,leaving behind the brightest (i.e., best producing) clones for use inantibody production.

EXAMPLE 9

Automated Monitoring of Cellular Responses

Automated monitoring of cellular responses to specific stimuli is ofgreat interest in high-throughput drug screening. Often, a cellpopulation in one well of a well-plate is exposed to a stimulus, and afluorescent signal is then captured over time from the cell populationas a whole. Using the methods and apparatus described herein, moredetailed monitoring could be done at the single cell level. For example,a cell population can be labeled to identify a characteristic of asubpopulation of cells that are of interest. This label is then excitedby the illumination laser to identify those cells. Thereafter, thetreatment laser is targeted at the individual cells identified by thefirst label, for the purpose of exciting a second label, therebyproviding information about each cell's response. Since the cells aresubstantially stationary on a surface, each cell could be evaluated ortreated multiple times, thereby providing temporal information about thekinetics of each cell's response. Also, through the use of the largearea scanning lens and galvanometer mirrors, a relatively large numberof wells could be quickly monitored over a short period of time.

As a specific example, consider the case of alloreactive T-cells aspresented in Example 2 above. In the presence of allo-antigen, activateddonor T-cells could be identified by CD69. Instead of using thetreatment laser to target and kill these cells, the treatment lasercould be used to examine the intracellular pH of every activated T-cellthrough the excitation and emitted fluorescence of carboxyfluoresceindiacetate. The targeted laser allows the examination of only cells thatare activated, whereas most screening methods evaluate the response ofan entire cell population. If a series of such wells are being monitoredin parallel, various agents could be added to individual wells, and thespecific activated T-cell response to each agent could be monitored overtime. Such an apparatus would provide a high-throughput screening methodfor agents that ameliorate the alloreactive T-cell response ingraft-versus-host disease. Based on this example, one skilled in the artcould imagine many other examples in which a cellular response to astimulus is monitored on an individual cell basis, focusing only oncells of interest identified by the first label.

Although aspects of the present invention have been described byparticular embodiments exemplified herein, the present invention is notso limited. The present invention is only limited by the claims appendedbelow.

We claim:
 1. A method for inducing a response in one or more targetedcells within a biological specimen, comprising: illuminating a frame ofcells in said biological specimen with a first wavelength of light,wherein said biological specimen has been treated with more than onelabel that, when excited by said first wavelength of light, emit energyat one or more different wavelengths; capturing an image of said frameof cells; determining the locations of one or more targeted cells withinsaid image by reference to cells that are activated by said firstwavelength of light; and steering an energy beam to said locations ofsaid one or more targeted cells, wherein said energy beam is sufficientto induce a response in at least one of said one or more targeted cells.2. The method of claim 1, wherein determining said locations of said oneor more targeted cells comprises determining the presence of, absenceof, or relative intensity of said more than one label attached to saidone or more targeted cells.
 3. The method of claim 1, wherein capturingsaid one or more images of said frame of cells comprises capturing oneor more images of labeled cells.
 4. The method of claim 1, wherein saidresponse is selected from the group consisting of: cell death, celllysis, photomechanical disruption, photoablation, optoporation,activation of a photosensitive agent, inactivation of a cell component,controlled movement, and excitation of a fluorescent reagent.
 5. Amethod for inducing a first response in a first targeted population ofcells and a second response in a second targeted population of cells,comprising: illuminating said first targeted population of cells andsaid second targeted population of cells with a first wavelength oflight; capturing an image comprising said illuminated first targetedpopulation of cells and said illuminated second targeted population ofcells; determining the locations of cells of said first targetedpopulation of cells by reference to said image; determining thelocations of said second targeted population of cells by reference tosaid image; and steering an energy beam to said locations of said cellsof said first targeted population of cells and said cells of said secondtargeted population of cells in order to induce said first response insaid cells of said first targeted population of cells and said secondresponse in said cells of said second targeted population of cells. 6.The method of claim 5, wherein determining said locations of said cellsof said first targeted population of cells comprises determining thepresence of, absence of, or relative intensity of one or more labelsthat are attached to said cells of said first targeted population ofcells.
 7. The method of claim 5, wherein determining said locations ofsaid cells of said second targeted population of cells comprisesdetermining the presence of, absence of, or relative intensity of one ormore labels that are attached to said cells of said second targetedpopulation of cells.
 8. The method of claim 5, wherein said firstresponse and said second response are selected from the group consistingof: cell death, cell lysis, photomechanical disruption, photoablation,optoporation, activation of a photosensitive agent, inactivation of acell component, controlled movement, and excitation of a fluorescentreagent.